The Snf1 protein kinase plays a central role in nutrient stress signaling in the budding yeast, Saccharomyces cerevisiae. The mammalian homologue of Snf1, the AMP-activated protein kinase (AMPK) plays an equally important role in mammalian cells. Recently AMPK was shown to be the target of metformin, the most widely prescribed drug used to treat type 2 diabetes. We propose to study the regulation of this medically important enzyme in yeast. In the previous grant period we developed an assay for the phosphorylation state of the Snf1 activation loop. We showed that phosphorylation of Snf1 was a critical step for the activation of the Snf1 kinase and that the phosphorylation of Snf1 was catalyzed by a distinct upstream kinase. Recently, we were able to demonstrate that there are three upstream kinases (Pakl, Tos3 and Elm1) that are capable of activating Snf1 in vivo. The identification of the Snf1 activating kinases was highly significant because it immediately led to the identification of the mammalian kinase that activates AMPK. In the next grant period we propose to continue our studies of the regulation of the Snf1 kinase signaling pathway. We will focus on upstream events in this pathway and determine the mechanism that regulate the activity of the three Snf1-activating kinases. We will determine whether there are distinct forms of the Snf1 kinase complex that are specialized for response to different stresses. We will assess the regulatory significance of phosphorylation sites in the Snf1 kinase complex that we have mapped by mass spectrometry. Finally, biochemical and genetic analyses have identified a number of candidates for regulators of the Snf1 kinase signaling pathway. These candidates proteins will be studied to determine if they associate with and regulate the activity of the Snf1 kinase.